Peripheral sympathetic nerves co‐release multiple neurotransmitters to modulate the activity of their target organ, for example mediating physiological regulation of cardiac contractility and systemic vascular tone. However, the release of “co‐released” transmitters like norepinephrine (NE) and ATP can be differentially regulated, for example by relying on specific isoforms of voltage‐gated Ca2+ channels (CaV), and we previously reported that NE‐ and ATP‐containing vesicles segregate in pools that preferentially co‐localize with specific CaV2 isoforms in rat tail artery. Whether this observation generalizes to other sympathetic nerves is not known, but it is of fundamental importance to understanding the molecular biology of sympathetic co‐release. OBJECTIVE To determine whether specific isoforms of CaV selectively associate with pools of vesicles enriched with transporters for NE or ATP in multiple peripheral sympathetic nerves. METHODS We performed epifluorescence imaging of intact rat tail artery and cultured superior cervical ganglia (SCG) neurons, comparing the localization and intensity correlation of CaV2.1, CaV2.2, and CaV2.3 with vesicle markers for NE (VMAT2) and ATP (VNUT/SlC17A) containing vesicles or suitable surrogate markers. RESULTS We confirmed that VMAT2 and VNUT are often but not always found in the same varicosities. While VMAT2 is broadly distributed in varicosity, VNUT localizes as discrete puncta at the perimeter of varicosity and does not co‐localize with synaptagmin1 in SCG neurons, similar rat artery nerves. CaV2.3 and CaV2.1, like VNUT, localize to the periphery of the varicosities, while CaV2.2 more closely mimics to the distribution of VMAT2. These findings mirror our observations in rat tail artery, in which NE release is most sensitive to CaV2.2 inhibition and ATP‐mediated contraction is blocked by CaV2.3 inhibition. CONCLUSION We show that synaptic organization of CaV2 isoforms with specific pools of vesicle in a cultured nerve system closely recapitulates that found in vivo. This observation further validates cultured SCG neurons as an experimentally tractable and physiologically revenant model to study presynaptic cell biology relevant to the regulation of both cardiac output and vasoregulation. Support or Funding Information 1. Natural Sciences and Engineering Research Council of Canada.2. Canadian Foundation for Innovation
ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.
Absolute quantification of mitochondrial DNA copy number (mCN) provides important insights in many fields of research including cancer, cardiovascular and reproductive health. Droplet digital PCR (ddPCR) natively reports absolute copy number, and we have developed a single-dye, multiplex assay to measure rat mCN that is accurate, precise and affordable. We demonstrate simple methods to optimize this assay and to determine nuclear reference pseudogene copy number to extend the range of mCN that can be measured with this assay. We evaluated two commonly used mitochondrial DNA reference loci to determine mCN, the ND1 gene and the D-Loop. Harnessing the absolute measures of ddPCR, we found that the D-Loop amplifies with a copy number of ~1.0-1.5 relative to other sites on the mitochondrial genome. This anomalous copy number varied significantly between rats and tissues (aorta, brain, heart, liver, soleus muscle). We advocate for avoiding the D-Loop as a mitochondrial reference in future studies of mCN. Further, we report a novel approach to quantifying immunolabelled mitochondrial DNA that provides single-cell estimates of mCN that closely agree with the population analyses by ddPCR. The combination of these assays represents a cost-effective and powerful suite of tools to study mCN.
Background We previously reported elevated hypertension (HT) rates in adults with the m.3243A>G mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes (MELAS) mutation. The cause of HT in MELAS is not known but could be linked to dysautonomia and endothelial dysfunction. Hypertension is also reported in transgenic mice that have an error‐prone gamma DNA polymerase (POLG) that increases mtDNA mutation rate. In contrast to MELAS, HT in POLG−/− mice appears to be due to hypertrophy of the tunica media . It is not known if humans with pathogenic POLG mutations similarly exhibit HT, and whether the hemodynamic basis of HT in MELAS and POLG are consistent with common or distinct pathogenic mechanisms. Purpose We aimed to examine whether POLG patients have an increased risk of HT, and whether the hemodynamics and therapeutic sensitivity of such HT resembles that of HT in MELAS patients. Specifically, we hypothesized that POLG patients would be prone to isolated systolic HT, while MELAS patients should exhibit remodeling of resistance vessels. Methods We conducted a chart review at the Adult Metabolic Disease Clinic, Vancouver General Hospital of 35 MELAS and 26 POLG patients in accordance with institutional human research ethics guidelines. Demographic factors, including age, weight, BMI, sex, smoking status and diabetes status were collected. Patients were scored as hypertensive if they were on anti‐hypertensive drugs or had consistently elevated blood pressure measurements in accordance with the Canadian Hypertension Education Program Guidelines. Results MELAS and POLG patients showed increased prevalence of HT (20–39 yr age group: 30% MELAS, 14% POLG; 40–59 yr: 71%, 57%; 60–79 yr: 83%, 81%) compared to the Canadian population. Hypertension was not associated with sex, smoking or BMI, and exceeded expected levels given 50% of MELAS patients were diabetic. In the 40–59 and 60–79 yr age groups, antihypertensive drugs controlled systolic blood pressure in MELAS but not in POLG patients (40–59 yr: POLG 145 ± 21 mmHg n = 8 vs MELAS 122 ± 23 n = 12; 60–79 yr: POLG 157 ± 6 n=4 vs MELAS 134 ± 21 n=5), while diastolic blood pressure did not differ between MELAS and POLG. Preliminary analyses suggest that MELAS, but not POLG, patients exhibit elevated peripheral resistance despite therapeutic correction of blood pressure. Conclusions MELAS and POLG increase the risk of HT, which can increase the propensity for cardiac hypertrophy and cerebrovascular problems. Signs of vascular remodeling as a contributor to HT suggest that early therapeutic treatment at the onset of pre‐hypertension may be warranted to prevent and control HT in these diseases, especially POLG. These findings warrant detailed hemodynamic assessments in larger populations.
During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca 2+ -mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.
1 We characterized the mechanisms in vascular smooth muscle cells (VSMCs) that produce asynchronous, wave-like Ca2+ oscillations in response to phenylephrine (PE). Confocal imaging was used to observe [Ca2+]i in individual VSMCs of intact inferior vena cava (IVC) from rabbits. 2 It was found that the Ca2+ waves were initiated by Ca2+ release from the sarcoplasmic reticulum (SR) via inositol 1,4,5-trisphosphate-sensitive SR Ca2+ release channels (IP3R channels) and that refilling of the SR Ca2+ store through the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) was required for maintained generation of the repetitive Ca2+ waves. 3 Blockade of L-type voltage-gated Ca2+ channels (L-type VGCCs) with nifedipine reduced the frequency of PE-stimulated [Ca2+]i oscillations, while additional blockade of receptor-operated channels/store-operated channels (ROCs/SOCs) with SKF96365 abolished the remaining oscillations. Parallel force measurements showed that nifedipine inhibited PE-induced tonic contraction by 27% while SKF96365 abolished it. This indicates that stimulated Ca2+ entry refills the SR to support the recurrent waves of SR Ca2+ release and that both L-type VGCCs and ROCs/SOCs contribute to this process. 4 Application of the Na+-Ca2+ exchanger (NCX) inhibitors 2′,4′-dichlorobenzamil (forward- and reverse-mode inhibitor) and KB-R7943 (reverse-mode inhibitor) completely abolished the nifedipine-resistant component of [Ca2+]i oscillations and markedly reduced PE-induced tone. 5 Thus, we conclude that each Ca2+ wave depends on initial SR Ca2+ release via IP3R channels followed by SR Ca2+ refilling through SERCA. Na+ entry through ROCs/SOCs facilitates Ca2+ entry through the NCX operating in the reverse mode, which refills the SR and maintains PE-induced [Ca2+]i oscillations. In addition some Ca2+ entry through L-type VGCCs and ROCs/SOCs serves to modulate the frequency of the oscillations and the magnitude of force development.
Amiloride derivatives are blockers of the Na(+)/H(+) exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action.We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca(2+) concentration ([Ca(2+)](ER)) with a 'cameleon' indicator, D1ER.Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca(2+)](ER), mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca(2+)](ER). The decrease in [Ca(2+)](ER) evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cytosolic Ca(2+) concentration.Amiloride derivatives disrupt ER and cytosolic Ca(2+) homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca(2+) depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives.
